Hydrogel materials are used in culturing tasks where hydrophilic supporting material is needed, for example agar type hydrocolloids are widely used in plant cell, bacterial, and fungi culturing for various microbiological purposes.
Agar is a linear and non-ionic polysaccharide consisting of D-galactose and 3,6-anhydro-L-galactose and it is produced from seaweeds. In solid cultures suspensions of microbial cells are spread onto the surface of the agar hydrogel, typically containing 1.5 wt % of agar, and nutrient fluid. The microorganisms grow and form macroscopic colonies, which can be separated and pure cultures may be obtained. The use of solid agar plates provides two-dimensional growth and requires mechanical separation by cutting.
Several alternatives for the use of agar plates have been proposed, for example gellan gum produced by Pseudomonas elodea. Gellan gum is soluble in hot water, forms a stiff gel upon cooling and shows improved stability at higher temperatures. Hydrogels based on gellan gum are very sensitive to nutrients and additives and require careful formulation of the medium. Also the use of solid gellan gum plates provides two-dimensional growth.
In Deguchi, S. et al. Preparation and characterisation of nanofibrous cellulose plate as a new solid support for microbial culture, Soft Matter, 2007, Vol. 3, No. 9, s. 1170-1175 a nanofibrous cellulose plate is suggested for solid culture of microorganisms where the cellulose was obtained by dispersing microcrystalline cellulose in an aqueous saturated solution of Ca(SCN)2 to form a complex between cellulose and calcium thiocyanate ions, followed by dissolving the cellulose by heating, and obtaining a viscous solution. Said solution was then poured into a culture dish, allowed to solidify and followed by washing with methanol and water. After washing the gelation was fixed. The optimal cellulose concentration in the plates was between 2 and 3 wt %. The pores of the plates were filled with an appropriate nutrient fluid. E. coli, B. subtilis, and S. cerevisiae as well as T. thermophilus grew on the cellulose plates.
U.S. Pat. No. 5,254,471 discloses a carrier for culturing cells, made of ultra fine fibers. WO 2009/126980 discloses cellulose-based hydrogel, which contains cellulose exhibiting an average degree of polymerization of 150-6200.
Anaerobic and semi-anaerobic microbial culture techniques are typically regarded as challenging to perform with current growth media and plating systems. There are several microbe species having strong sensitivity to oxygen, such as Clostridial species, and thus it is essential to remove oxygen from the cultivation environment. Further, microbial colonies are typically cut from the culturing media and thereafter the culturing media has to be removed.
Anaerobic and semi-anaerobic microbes are often cultured in embedded systems. In said systems, for example paraffin embedded cultures of microbes have been proposed, as well as cultures between two agar layers or plates.
In connection with microbial culture, fermentation and microbial sample storage, detection, enumeration and quantification of microbes based on techniques where real-time polymerase chain reaction (PCR) is carried out, are today widely used. In PCR the microbes are broken down to release their DNA, and the DNA is thereafter quantified by using specific oligonucleotide primers, thermostable DNA polymerase and appropriate thermal cycler. Many materials, especially polymeric materials inhibit the PCR reactions and make microbial quantification unreliable. Typically such materials are used as fermentation media, culturing media, sample storage matrix, fermentation enhancers, and transportation matrix, which interfere with the detection and quantification procedures.
Existing three dimensional (3D) cell culture biomaterials do not allow transferring the hydrogel matrix for example with a needle without seriously damaging the cultured cells.
Thus there exists a need to provide improved compositions, matrix and methods for embedded microbial culture including anaerobic and semi-anaerobic methods, where the disadvantages of the materials of state of the art can be avoided or at least substantially decreased.